The present invention relates to novel thermo-stable DNA polymerases, the genes and vectors encoding them and their use in DNA sequencing.
U.S. Pat. Nos. 4,889,818 and 5,079,352 describe the isolation and expression of a DNA polymerase known as Taq DNA Polymerase (hereinafter referred to as Taq). It is reported that amino-terminal deletions wherein approximately one-third of the coding sequence is absent have resulted in producing a gene product that is quite active in polymerase assays. Taq is described as being of use in PCR (polymerase chain reaction).
U.S. Pat. No. 5,075,216 describes the use of Taq in DNA sequencing.
International patent application WO 92/06/06188 describes a DNA polymerase having an identical amino acid sequence to Taq except that it lacks the N-terminal 235 amino acids of Taq and its use in sequencing. This DNA polymerase is known as .DELTA. Taq.
U.S. Pat. No. 4,795,699 describes the use of T7 type DNA polymerases (T7) in DNA sequencing. These are of great use in DNA sequencing in that they incorporate dideoxy nucleoside triphosphates (NTPs) with an efficiency comparable to the incorporation of deoxy NTPs; other polymerases incorporate dideoxy NTPs far less efficiently which requires comparatively large quantities of these to be present in sequencing reactions.
At the DOE Contractor-Grantee Workshop (Nov. 13-17, 1994, Santa Fe) and the I. Robert Lehman Symposium (Nov. 11-14, 1994, Sonoma), Prof. S. Tabor identified a site in DNA polymerases that can be modified to incorporate dideoxy NTPs more efficiently. He reported that the presence or absence of a single hydroxy group (tyrosine vs. phenylalanine) at a highly conserved position on E. coli, DNA Polymerase I, T7, and Taq makes more than a 1000-fold difference in their ability to discriminate against dideoxy NTPs. (See also European Patent Application 94203433.1 published May 31, 1995, Publication No. 0 655 506 A1 and hereby incorporated by reference herein.)